The plasma membrane is believed to play an important role in the successful establishment of hematogenous metastases. The membrane bears 1) enzymes required for destruction of basement membrane barriers; 2) receptors for homotypic and heterotypic cell associations; and 3) antigens which direct specific and nonspecific immune reactivities. The purpose of this study is to isolate and characterize cells surface moieties that influence metastatic phenotype, using a combination of butanol extraction, preparative isoelectric focusing, high performance gel permeation, ion exchange and hydrophobic interaction chromatography, and lectin affinity chromatography. The applicant has employed noncytolytic extraction using butanol to enhance the incidence of experimentally-induced pulmonary metastases. Extraction increases the metastatic potential of sublines of either low or high metastatic phenotype derived from the chemically induced fibrosarcoma, MCA-F, or spontaneous melanoma, B16. Brief incubation of extracted cells in crude butanol extracts (CBE) or partially-purified preparations reconstitutes the metastatic phenotype to that of the unextracted controls. The reconstitutive activity in partially purified preparations is quantitatively assessed as the mg amount of protein necessary to reduce the incidence of experimentally-induced pulmonary metastases by 50%. The reconstituting activity from B16-F1 displays of pI of 546 to 5.8, a mol. wt of 15 to 30 kDa, and a specific reconstituting activity of 10-4 U/mg. The mechanism of reconstitution is also under investigation. We are currently testing the hypothesis that extraction removes cell surface-associated protease inhibitors, resulting in a facilitated egress of pulmonary emboli into the surrounding tissues. Immunization of syngeneic hosts with CBE from B16-F10 prior to i.v. challenge significantly increases the incidence of experimentally-induced lung metastases, and this effect can be adoptively transferred using spleen cells. Both adherent and nonadherent splenic populations are necessary to transfer suppression. The final objective of this proposal is to delineate the cellular immune response to suppressogens in CBE, and to characterize the molecules and immune cells participating. Thus, butanol extraction of metastatic cells allows investigation of possible mechanisms involved in hematogenous metastases, the role of tumor cell surface structures in metastasis, and the effect of antigenic stimulation on the eventual development of metastases.